Emergence of highly pathogenic avian influenza A(H5N8)clade 2.3.4.4b viruses in grebes in Inner Mongolia and Ningxia,China,in 2021

Highly pathogenic avian influenza(HPAI)subtype H5Nx viruses have spread globally and are a major concern for poultry,wild birds,mammals,and even humans(de Vries et al.2015;Zeng et al.2022).The hemagglutinin(HA)genes of H5 subtype viruses have evolved into multiple clades and some of these clades have been further divided into subclades(Cui et al.2022).Clade 2.3.4.4H5N8 HPAI viruses(HPAIVs)have caused several waves of disease outbreaks in wild birds and domestic poultry(Wang et al.2022).

Generation and application of two monoclonal antibodies targeting conserved linear epitopes in the NP protein of influenza A virus

Monoclonal antibodies(mAbs) are widely used in virus research and disease diagnosis. The nucleoprotein(NP) of influenza A virus(IAV) plays important roles in multiple stages of the virus life cycle. Therefore, generating conserved mAbs against NP and characterizing their properties will provide useful tools for IAV research. In this study, two mAbs against the NP protein, 10 E9 and 3 F3, were generated with recombinant truncated NP proteins(NP-1 and NP-2) as immunogens. The heavy-chain subclass of both 10 E9 and 3 F3 was determined to be IgG2α, and the light-chain type was κ. Truncation and site-specific mutation analyses showed that the epitopes of mAbs 10 E9 and 3 F3 were located in the N terminal 84–89 amino acids and the C terminal 320–324 amino acids of the NP protein, respectively. We found that mAbs 10 E9 and 3 F3 reacted well with the NP protein of H1–H15 subtypes of IAV. Both 10 E9 and 3 F3 can be used in immunoprecipitation assay, and 10 E9 was also successfully applied in confocal microscopy. Furthermore, we found that the 10 E9-recognized _(84) SAGKDP_(89) epitope and 3 F3-recognized 320 ENPAH324 epitope were highly conserved in NP among all avian and human IAVs. Thus, the two mAbs we developed could be used as powerful tools in the development of diagnostic methods of IAV, and also surely promote the basic research in understanding the replication mechanisms of IAV.

Co-infection of SARS-COV-2 and Influenza A Virus:A Case Series and Fast Review

Coronavirus disease 2019(COVID-19)occurs in the influenza season and has become a global pandemic.The present study aimed to examine severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)co-infection with influenza A virus(IAV)in an attempt to provide clues for the antiviral interventions of co-infected patients.We described two patients who were co-infected with SARS-CoV-2 and IAV treated at Wuhan Union Hospital,China.In addition,we performed a review in PubMed,Web of Science and CNKI(from January 1 up to November 1,2020)with combinations of the following key words:“COVID-19,SARS-COV-2,influenza A and co-infection”.A total of 28 co-infected patients were enrolled in the analysis.Of the 28 patients,the median age was 54.5 years(IQR,34.25–67.5)and 14 cases(50.0%)were classified as severe types.The most common symptoms were fever(85.71%),cough(82.14%)and dyspnea(60.71%).Sixteen patients had lymphocytopenia on admission and 23 patients exhibited abnormal radiological changes.The median time from symptom onset to hospital admission was 4 days(IQR,3–6),and the median time of hospital stay was 14 days(IQR,8.5–16.75).In conclusion,patients with SARSCOV-2 and IAV co-infection were similar to those infected with SARS-COV-2 alone in symptoms and radiological images.SARS-COV-2 co-infection with IAV could lead to more severe clinical condition but did not experience longer hospital stay compared with patients infected with SARSCOV-2 alone.

Comparison of clinical characteristics in patients with coronavirus disease and influenza A in Guangzhou, China

BACKGROUND: This study aims to compare the epidemiological, clinical and laboratory characteristics between patients with coronavirus disease(COVID-19) and influenza A(H1N1), and to develop a differentiating model and a simple scoring system.METHODS: We retrospectively analyzed the data from patients with COVID-19 and H1N1. The logistic regression model based on clinical and laboratory characteristics was constructed to distinguish COVID-19 from H1N1. Scores were assigned to each of independent discrimination factors based on their odds ratios. The performance of the prediction model and scoring system was assessed. RESULTS: A total of 236 patients were recruited, including 20 COVID-19 patients and 216 H1N1 patients. Logistic regression revealed that age >34 years, temperature ≤37.5℃, no sputum or myalgia, lymphocyte ratio ≥20% and creatine kinase-myocardial band isoenzyme(CK-MB) >9.7 U/L were independent differentiating factors for COVID-19. The area under curves(AUCs) of the prediction model and scoring system in differentiating COVID-19 from H1N1 were 0.988 and 0.962, respectively. CONCLUSIONS: There are certain differences in clinical and laboratory features between patients with COVID-19 and H1N1. The simple scoring system may be a useful tool for the early identification of COVID-19 patients from H1N1 patients.

Integrated analysis of human influenza A(H1N1)virus infectionrelated genes to construct a suitable diagnostic model

The genome characteristics and structural functions of coding proteins correlate with the genetic diversity of the H1N1 virus,which aids in the understanding of its underlying pathogenic mechanism.In this study,analyses of the characteristic of the H1N1 virus infection-related genes,their biological functions,and infection-related reversal drugs were performed.Additionally,we used multi-dimensional bioinformatics analysis to identify the key genes and then used these to construct a diagnostic model for the H1N1 virus infection.There was a total of 169 differently expressed genes in the samples between 21 h before infection and 77 h after infection.They were used during the protein-protein interaction(PPI)analysis,and we obtained a total of 1725 interacting genes.Then,we performed a weighted gene co-expression network analysis(WGCNA)on these genes,and we identified three modules that showed significant potential for the diagnosis of the H1N1 virus infection.These modules contained 60 genes,and they were used to construct this diagnostic model,which showed an effective prediction value.Besides,these 60 genes were involved in the biological functions of this infectious virus,like the cellular response to type I interferon and in the negative regulation of the viral life cycle.However,20 genes showed an upregulated expression as the infection progressed.Other 36 upregulated genes were used to examine the relationship between genes,human influenza A virus,and infection-related reversal drugs.This study revealed numerous important reversal drug molecules on the H1N1 virus.They included rimantadine,interferons,and shikimic acid.Our study provided a novel method to analyze the characteristic of different genes and explore their corresponding biological function during the infection caused by the H1N1 virus.This diagnostic model,which comprises 60 genes,shows that a significant predictive value can be the potential biomarker for the diagnosis of the H1N1 virus infection.

Knowledge and preventive measures practiced by junior high school students from Mexico City regarding influenza A (H1N1)

Background: Influenza A (H1N1) is the most recent pandemic disease that has affected the human population. Objective: To evaluate knowledge and preventive measures related with this disease one year after the epidemic of Mexico- that took place in 2009. Material and methods: An epidemiologic survey regarding influenza A (H1N1) was conducted in June 2010 among 2541 students from the second grade of all public high schools in a borough in Mexico City. The questionnaire included items on the knowledge of the disease and practice of preventive measures. Results: Most students obtained the information from television, half of them from parents and only one fifth from teachers;72% of the participants had a favorable knowledge about the disease and the measures to avoid being infected. However, only 37% practiced such preventive measures. Conclusion: Knowledge has a positive influence on practices towards health. Parents and teachers have an important role in health education, thus efforts should be directed to involve them more intensely in health education.

Feedforward loop profile among transcription factor, miRNA and mRNA in influenza A virus-infected mouse lungs

Purpose:In the present study,we focused on the 46 microRNAs and 719 genes in the microRNA-gene network,reported by us,and aimed to build a research blueprint of feedforward loops and reveal the key TFs in H1N1-infected mouse lung.Method:Based on microRNAs and genes in the microRNA-gene network previously reported by us,we used Jemboss software to find relationships between TFs and microRNAs(or genes),and then built a TF-microRNA-gene network exploiting the interactions between TFs and microRNAs(or genes).Next,we searched the sequences of above genes or microRNAs near the transcription start site(TSS)area,and then used the MatchTM algorithm to predict relevant TFs,and built the TF-Gene-Network.Result:We built a TF-microRNAgene network and exploreed eight key TFs,namely NF-AT1,GKLF,SRY,SOX10,AML1,MZF1,CRX and myogenin,in the network,and then constructed subgraphs of these eight TFs.Simultaneously,we predicted the possible target genes of microRNAs and identified the feedforward regulation relationship of possible TFs,microRNAs and mRNAs.The results showed that all eight factors with a score greater than 100 were TFs,namely NF-AT1,GKLF,SRY,SOX10,AML1,CRX,myogenin and MZF1.We then constructed subtables of the above eight TFs.Conclusion:In this study,TFs including NF-AT1,GKLF,SRY,SOX10,AML1,MZF1,CRX and myogenin showed the highest score(>100)not only in the TF-microRNA-gene network but also in feedforward loops,indicating that these eight TFs play the most important roles in mouse H1N1 influenza virus infection biology.

Study on the mechanism of action of Ephedra Herba Decoction against influenza A virus based on network pharmacology

Objective To predict the main bioactive components and potential mechanisms of Ephedra Herba Decoction(Chinese Pinyin abbreviated as MHT)in treating influenza A virus(IAV)based on network pharmacology.Methods Multiple online databases were used to search and screen out the active components from MHT,the related targets of active components of MHT and the genes related to IAV.Search the corresponding genes name of target through UniProt database.Cytoscape 3.7.2 was used to construct the drug-component-target network diagram.Venn diagram was used to screen the intersection genes of the active components corresponding to the target and disease-related genes,and the intersection genes were imported into the STRING Database Online platform to obtain the protein-protein interaction(PPI)network.Then,the PPI network was imported into Cytoscape 3.7.2 to obtain the core targets.Finally,Wei Sheng Xing(http://www.bioinformatics.com.cn/)was used to do GO function enrichment analysis and KEGG signaling pathway enrichment analysis for the intersection genes,and the results of GO and KEGG were visualized.Results A total of 116 active components and 253 potential targets were screened from MHT.Quercetin,Kaempferol,and Luteolin are the main active components,and AKT1,TNF,TP53,IL6,and JUN are the core targets of 253 potential targets.There are 2906 targets of influenza A,including 121 intersection genes.Enrichment analysis showed that there were 131 entries of molecular function,89 entries of cell component,1645 entries of biological process and 195 entries of signaling pathway.PI3K-Akt,MAPK,and JAK-STAT were the main signaling pathways.Conclusion MHT plays an important role in the prevention and treatment of influenza A by acting on multiple targets and multiple signaling pathways.

Clinical Characteristics of H1N1 Influenza A-Associated Mild Encephalopathy with Reversible Splenial Lesion: 4 Pediatric Cases

Objective Mild encephalopathy with reversible splenial lesion(MERS)is associated with a variety of infections and anti-epileptic drug withdrawal.Here we report the clinical characteristics of H1N1 influenza A-associated MERS based on our experience of four pediatric cases.Methods A detailed retrospective analysis of four patients with H1N1 influenza A-associated MERS was performed at Guangzhou Women and Children’s Medical Center.Results All patients exhibited mild influenza-like illness and seizures.Three patients presented with a new-onset seizure with fever after 5 years of age.75%patients had altered mental status.For all four patients,influenza A(H1N1)viral RNA was detected in throat swab specimens at least twice.Brain magnetic resonance images revealed similar ovoid lesions in the corpus callosum,mainly in the splenium and for one patient in the splenium and genu of the corpus callosum.Only one patient had an abnormal electroencephalogram tracing.Cells and protein in the cerebrospinal fluid were normal in all patients.All patients received oseltamivir and one patient received intravenous immunoglobulin.As a result,all patients fully recovered after 2 months and showed no neurologic sequelae at discharge.Conclusion This case series provides insight towards clinical features of H1N1 influenza A-associated MERS.

Morphofunctional Characteristics of Pulmonary Surfactant System and Its Effect on Immune Cells in Influenza A (H1N1) Pathogenesis

There is an annual increase of influenza-related SARI cases in winter months. Despite the high relevance of this problem, influenza pathogenesis and the role of surfactant system and its SP-A (surfactant protein A) enzyme in antiviral defense remain poorly understood. SP-A activates macrophage M1 polarization and triggers an antiviral response due to the activation of T-cells and dendritic cells. Therefore, surfactant system is an important element of infection protection and a promising therapeutic target.

Seroprevalence study of infection with influenza A(H1N1)pdm09 virus in San Felipe Town, Chile

Objective: To know the natural history of the first wave of pandemic influenza A(H1N1)pdm09 in the Southern hemisphere, through the detection of antibodies against influenza A(H1N1)pdm09 in a selected community, to estimate the population attack rate and its variations, the consultation rates, hospitallization and mortality rates. Methodology: A representative random sample of the population of a commune in Chile (San Felipe) was interviewed and taken blood samples between January and March 2010. A study against the antibodies of the influenza A(H1N1) pdm09 virus was conducted, by the technique of the Hemaglutination Inhibition (HAI) according to standardized methodology. Subjects with antibody titers ≥1:40 were considered positive. Results: 13.5% of the population of San Felipe had antibodies against influenza A(H1N1)pdm09;this percentage reached 30% of the population between 0 and 18 years and 6.1% among those over 19 years. The age variable was the only factor that evidenced significant differences in the prevalence of antibodies. There were no significant differences related to gender, vaccination history against seasonal inluenza, or comorbidity. 51% of people with positive serology showed IN-FLUENZA-LIKE SYMPTOMS. Conclusions: A relevant percentage of subclinical disease was detected in the first pandemic wave in Chile and the proportion of people with SARI and deaths was small. Data from epidemiological surveillance were useful to estimate the trend of TSI but not its magnitude.

Erucic acid from Isatis indigotica Fort. suppresses influenza A virus replication and inflammation in vitro and in vivo through modulation of NF-kB and p38 MAPK pathway

Isatis indigotica Fort.(Ban-Lan-Gen)is an herbal medicine prescribed for influenza treatment.However,its active components and mode of action remain mostly unknown.In the present study,erucic acid was isolated from Isatis indigotica Fort.,and subsequently its underlying mechanism against influenza A virus(IAV)infection was investigated in vitro and in vivo.Our results demonstrated that erucic acid exhibited broad-spectrum antiviral activity against IAV resulting from reduction of viral polymerase transcription activity.Erucic acid was found to exert inhibitory effects on IAV or viral(v)RNA-induced pro-inflam-matory mediators as well as interferons(IFNs).The molecular mechanism by which erucic acid with antiviral and anti-inflammatory properties was attributed to inactivation of NF-kB and p38 MAPK signaling.Furthermore,the NF-kB and p38 MAPK inhibitory effect of erucic acid led to diminishing the transcriptional activity of interferon-stimulated gene factor 3(ISGF-3),and thereby reducing IAV-triggered pro-inflammatory response amplification in IFN-β-sensitized cells.Additionally,IAV-or vRNA-triggered apoptosis of alveolar epithelial A549 cells was prevented by erucic acid.In vivo,erucic acid administration consistently displayed decreased lung viral load and viral antigens expression.Meanwhile,erucic acid markedly reduced CD8+cytotoxic T lymphocyte(CTL)recruitment,pro-apoptotic signaling,hyperactivity of multiple signaling pathways,and exacerbated immune inflammation in the lung,which resulted in decreased lung injury and mortality in mice with a mouse-adapted A/FM/1/47-MA(H1N1)strain infection.Our findings provided a mechanistic basis for the action of erucic acid against IAV-mediated inflammation and injury,suggesting that erucic acid may have a therapeutic potential in the treatment of influenza.

The interaction between the 2009 H1N1 influenza A hemagglutinin and neuraminidase: mutations, co-mutations, and the NA stalk motifs

As the world is closely watching the current 2009 H1N1 pandemic unfold, there is a great interest and need in understanding its origin, genetic structures, virulence, and pathogenicity. The two surface proteins, hemagglutinin (HA) and neuraminidase (NA), of the influenza virus have been the focus of most flu research due to their crucial biological functions. In our previous study on 2009 H1N1, three aspects of NA were investigated: the mutations and co-mutations, the stalk motifs, and the phylogenetic analysis. In this study, we turned our attention to HA and the interaction between HA and NA. The 118 mutations of 2009 H1N1 HA were found and mapped to the 3D homology model of H1, and the mutations on the five epitope regions on H1 were identified. This information is essential for developing new drugs and vaccine. The distinct response patterns of HA to the changes of NA stalk motifs were discovered, illustrating the functional dependence between HA and NA. With help from our previous results, two co-mutation networks were uncovered, one in HA and one in NA, where each mutation in one network co-mutates with the mutations in the other network across the two proteins HA and NA. These two networks residing in HA and NA separately may provide a functional linkage between the mutations that can impact the drug binding sites in NA and those that can affect the host immune response or vaccine efficacy in HA. Our findings demonstrated the value of conducting timely analysis on the 2009 H1N1 virus and of the integrated approach to studying both surface proteins HA and NA together to reveal their interdependence, which could not be accomplished by studying them individually.

Trends in global warming and evolution of polymerase basic protein 2 family from influenza a virus

Both global warming and influenza trouble humans in varying ways, therefore it is important to study the trends in both global warming and evolution of influenza A virus, in particular, proteins from influenza A virus. Recently, we have conducted two studies along this line to determine the trends between global warming and polymerase acidic protein as well as matrix protein 2. Although these two studies reveal some interesting findings, many studies are still in need because at least there are ten different proteins in influenza A virus. In this study, we analyze the trends in global warming and evolution of polymerase basic protein 2 (PB2) from influenza A virus. The PB2 evolution from 1956 to 2008 was defined using the unpredictable portion of aminoacid pair. Then the trend in this evolution was compared with the trend in the global temperature, the temperature in north and south hemispheres, and the temperature in influenza A virus sampling site and species carrying influenza A virus. The results show the similar trends in global warming and in PB2 evolution, which are in good agreement with our previous studies in polymerase acidic protein and matrix protein 2 from influenza A virus.

Cross-neutralizing Anti-hemagglutinin Antibodies Isolated from Patients Infected with Avian Influenza A(H5N1) Virus

Objective To recover broad-neutralizing monoclonal antibodies(Bn Abs)from avian influenza A(H5N1)virus infection cases and investigate their genetic and functional features.Methods We screened the Abs repertoires of expanded B cells circulating in the peripheral blood of H5N1 patients.The genetic basis,biological functions,and epitopes of the obtained Bn Abs were assessed and modeled.Results Two Bn Abs,2-12 D5,and 3-37 G7.1,were respectively obtained from two human H5N1 cases on days 12 and 21 after disease onset.Both Abs demonstrated cross-neutralizing and Ab-dependent cellular cytotoxicity(ADCC)activity.Albeit derived from distinct Ab lineages,i.e.,V^H1-69-D2-15-JH^4(2-12D5)and V^H1-2-D3-9-JH^5(3-32 G7.1),the Bn Abs were directed toward CR6261-like epitopes in the HA stem,and HA2 I45 in the hydrophobic pocket was the critical residue for their binding.Signature motifs for binding with the HA stem,namely,IFY in VH1-69-encoded Abs and LXYFXW in D3-9-encoded Abs,were also observed in 2-12D5 and 3-32 G7.1,respectively.Conclusions Cross-reactive B cells of different germline origins could be activated and re-circulated by avian influenza virus.The HA stem epitopes targeted by the Bn Abs,and the two Ab-encoding genes usage implied the VH1-69 and D3-9 are the ideal candidates triggered by influenza virus for vaccine development.

Spatio-temporal Differentiation in the Incidence of Influenza and Its Relationship with Air Pollution in China from 2004 to 2017

The Healthy China Initiative is a major health strategy being pursued by the country.To prevent and control different types of diseases as well as their complex variants,research on the spatio-temporal differentiation among and mechanisms of influence of epidemic diseases is growing worldwide.This study analyzed monthly data on the incidence of influenza by using different methods,including Moran’s I,the hotspot analysis model,concentration analysis,and correlation analysis,to determine the characteristics of spatiotemporal differentiation in the incidence of influenza across prefecture-level cities in China from 2004 to 2017,and to examine its relationship with air pollution.According to the results,the overall incidence of influenza in China exhibited a trend of increase from 2004 to 2017,with small peaks in 2009 and 2014.More cases of influenza were recorded in the first and fourth quarters of each year.Regions with higher incidences of influenza were concentrated in northwestern and northern China,and in the coastal areas of southeastern China.Over time,the distribution of regions with a higher incidence of influenza has shifted from the west to the east of the country.A significant relationship was observed between the incidence of influenza and factors related to air pollution.The contents of five air pollutants(PM_(2.5),PM10,SO_(2),NO_(2),and CO)were significantly positively correlated with the incidence of influenza,with a decreasing order of contribution to it of SO_(2)>CO>NO_(2)>PM_(2.5)>PM_(10).The content of O_(3) in the air was negatively correlated with the incidence of influenza.The influence of air pollution-related factors on the incidence of influenza in different regions and seasons showed minor differences.The large-scale empirical results provided here can supply a scientific basis for governmental disease control authorities to formulate strategies for regional prevention and control.

Prediction of mutation position, mutated amino acid and timing in hemagglutinins from North America H1 influenza A virus

This study was trying to predict the mutations in H1 hemagglutinins of influenza A virus from North America including the predictions of mu-tation position, the predictions of would-be-mutated amino acids and the predictions of time of occurrence of mutations. The results paved a possible way for accurate, precise and reliable prediction of mutation in proteins from influenza A virus.

Knowledge,attitudes and practices towards avian influenza A(H5N1) among Cambodian women:A cross-sectional study

Objective:To measure highly pathogenic avian influenza(HPAI)-related knowledge,attitudes, and practices(KAPs) among Cambodian women.Methods:This cross-sectional study selected 246 married women aged between 18-55 years who had backyard poultry and lived at least one year in the areas of the survey through multi-stage cluster sampling.An average score of correct answers was generated to evaluate respondents’ knowledge(Good/Poor).attitudes(Positive/ Negative),and practices(Good/Bad).Results:We reported that about half of the respondents had good knowledge and good practices and four-fifth of them had positive attitudes towards HPAl.Odds ratios(ORs) and 95%confidence intervals(CIs) were estimated through a logistic regression model to explore contributing factors that raise their KAP levels.Most of the sources were significant in increasing knowledge of the respondent,like television(OR=l.6.95%CI=1.0- 2.7),radio(OR=2.5.95%CI=l.3-4.9).leaflets/booklets(OR=2.1,95%CI=1.2-3.9),school students (OR=18.4.95%CI=2.4-142.9).village health volunteers(OR=4.5.95%CI=2.2-10.9) etc.Factors such as television(OR=3.7.95%CI=2.1-6.4).leaflets/booklets(OR=2.6,95%CI7=1.4-5.1).and public health staff(OR=2.2,95%CI=1.2-4.1) had similar influence on practices.Although,we found similar effect on raising the attitudes of the responded,it was not significant.Conclusions: We report a satisfactory level of positive attitudes,and moderate level of knowledge and practices related to HPAI among Cambodian women.Raising KAPs through television,radio and other medias may be more efficient than using usual information,education and communication materials to prevent HPAI.

Novel H1N1 influenza A virus infection in a patient with acute rejection after liver transplantation

BACKGROUND:The 2009 H1N1 influenza A virus was first identified in April 2009 and rapidly evolved into a pandemic. Recipients of solid-organ transplants have a higher risk for severe infection because of immunosuppression.There are limited reports of 2009 H1N1 influenza in liver transplant recipients,especially in China. METHODS:We present a case of a 48-year-old male liver transplant recipient with 2009 H1N1 influenza A virus.He received therapy for acute rejection after transplantation and was confirmed with H1N1 virus infection. RESULTS:The patient was started on oseltamivir(75 mg, orally twice daily)and had a benign hospital course,with defervescence and resolution of symptoms within 72 hours. The follow-up chest radiograph after discharge was normal. CONCLUSIONS:The 2009 H1N1 influenza in this hospitalized transplant recipient was relatively mild,and prolonged viral shedding was not noted.Oseltamivir can be a valid measure in immunocompromised individuals.

Determination of inter- and intra-subtype/species varia-tions in polymerase acidic protein from influenza A virus using amino-acid pair predictability

The polymerase acidic protein is an important family of proteins from influenza A virus, which is classified as many different subtypes or spe-cies. Thus, an important question is if these classifications are numerically distinguishable with respect to the polymerase acidic protein. The amino-acid pair predictability was used to transfer 2432 polymerase acidic proteins into 2432 scalar data. The one-way ANOVA found these polymerase acidic proteins distinguish-able in terms of subtypes and species. However, the large residuals in ANOVA suggested a pos-sible large intra-subtype/species variation. Therefore, the inter- and intra-subtype/species variations were studied using the model II ANOVA. The results showed that the in-tra-subtype/species variations accounted most of variation, which was 100% in total for both inter- and intra- subtype/species variations. Our analysis threw lights on the issue of how to de-termine a wide variety of patterns of antigenic variation across space and time, and within and between subtypes as well as hosts.